The primary mechanism of terminating neurotransmission in the CMS is through active transport of neurotransmitters from the synaptic cleft. This process is mediated by integral membrane proteins located on the pre-synaptic neuron and surrounding glia. The superfamily of neurotransmitter sodium symporters (NSS) couples the transport of a variety of neurotransmitters into the cytoplasm to the movement of sodium ions down their concentration gradient. A major bottleneck in understanding the structure and dynamics of these transporters has been the lack of high-level expression systems and the availability of functional transporters in large quantities. The recent crystallization of a distant member of the superfamily, a functional bacterial Na+-dependent leucine transporter (LeuTAa), provided the first glimpse of transporter architecture and set the stage for an investigation of the detailed mechanism of transport. The long term goal of this research is to define the conformational motion that underlies Na+-dependent substrate binding and translocation using LeuTAaas a model. We will use spectroscopic techniques to obtain structural and dynamic constraints from defined intermediates putatively involved with transport. The specific aims will test the hypothesis that Na+ binding to LeuTAa opens the extracellular substrate permeation pathway and substrate binding alternatively exposes the binding site to the cytoplasm. Spin labels will be systematically introduced into the protein sequence and analyzed in both a detergent and lipid environment for mobility, solvent accessibility and inter-spin distances by electron paramagnetic resonance spectroscopy. The proposed work will provide a dynamic dimension to the structural analysis of a bacterial homologue of NSS. PUBLIC HEALTH RELEVANCE: Through their role in synaptic signaling, NSS play a critical role in complex physiological processes including movement, reward and mood. Understanding their mechanisms will provide a foundation for the interpretation of perturbing effects of illicit psychostimulants and pharmacological agents on these transporters. The sponsor is Hassanne Mchaourab, a Professor in the Department of Molecular Physiology and Biophysics at Vanderbilt. He has previously trained 2 PhD students and 5 postdoctoral fellows, and currently has 5 graduate students (including the applicant) and co-mentors two others. He has 2 R01s and a third with renewal pending, all on different projects.